Tumor necrosis factor-.alpha. (TNF.alpha.) is a cytokine which is released primarily by cells of immune systems in response to certain immunostimulators. When administered to animals or humans, it causes inflammation, fever, cardiovascular effects, hemorrhage, coagulation, cachexia, and acute phase responses similar to those seen during acute infections, inflammatory diseases, and shock states. Excessive or unregulated TNF.alpha. production has been implicated in a number of disease conditions. These include endotoxemia and/or toxic shock syndrome [Tracey, et al., Nature 330, 662-664 (1987) and Hinshaw, et al., Circ. Shock 30, 279-292 (1990)], rheumatoid arthritis, inflammatory bowel disease, cachexia [Dezube, et al., Lancet, 335 (8690), 662 (1990)], and lupus. TNF.alpha. concentration in excess of 12,000 pg/mL have been detected in pulmonary aspirates from Adult Respiratory Distress Syndrome (ARDS) patients [Millar, et al., Lancet 2(8665), 712-714 (1989)]. Systemic infusion of recombinant TNF.alpha. resulted in changes typically seen in ARDS [Ferrai-Baliviera, et al., Arch. Surg. 124(12), 1400-1405 (1989)].
TNF.alpha. appears to be involved in a number of bone resorption diseases, including arthritis. When activated, leukocytes will produce bone-resorption. TNF.alpha. apparently contributes to this mechanism. [Bertolini, et al., Nature 319, 516-518 (1986) and Johnson, et al., Endocrinology 124(3), 1424-1427 (1989)]. TNF.alpha. also has been shown to stimulate bone resorption and inhibit bone formation in vitro and in vivo through stimulation of osteoclast formation and activation combined with inhibition of osteoblast functions. Another compelling link with disease is the association between production of TNF.alpha. by tumor or host tissues and malignancy associated hypercalcemia [Calci. Tissue Int. (US) 46(Suppl.), S3-10 (1990)]. In Graft versus Host Reactions, increased serum TNF.alpha. levels have been associated with major complication following acute allogenic bone marrow transplants [Holler, et al., Blood, 75(4), 1011-1016 (1990)].
Validation of TNF-.alpha. inhibition as a clinical therapy has been demonstrated by the therapeutic use of TNF-.alpha. antibodies and soluble TNF-.alpha. receptors. TNF.alpha. blockage with monoclonal anti-TNF.alpha. antibodies has been shown to be beneficial in rheumatoid arthritis [Elliot, et al., Int. J. Pharmac. 1995 17(2), 141-145]. High levels of TNF.alpha. are associated with Crohn's disease [von Dullemen, et al., Gastroenterology, 1995 109(1), 129-135] treatment with soluble TNF.alpha. receptor treatment gave clinical benefits.
Cerebral malaria is a lethal hyperacute neurological syndrome associated with high blood levels of TNF.alpha. and the most severe complication occurring in malaria patients. Elevated levels of serum TNF.alpha. correlated directly with the severity of disease and the prognosis in patients with acute malaria attacks [Grau, et al., N. Engl. J. Med. 320(24), 1586-1591 (1989)].
TNF.alpha. plays a role in the area of chronic pulmonary inflammatory diseases. The deposition of silica particles leads to silicosis, a disease of progressive respiratory failure caused by a fibrotic reaction. Antibodies to TNF.alpha. completely blocked the silica induced lung fibrosis in mice [Pignet, et al., Nature, 344, 245-247 (1990)]. High levels of TNF.alpha. production (in the serum and in isolated macrophages) have been demonstrated in animal models of silica and asbestos induced fibrosis [Bissonnette, et al., Inflammation 13(3), 329-339 (1989)]. Alveolar macrophages from pulmonary sarcoidosis patients have also been found to spontaneously release massive quantities of TNF.alpha. as compared with macrophages from normal donors [Baughman, et al., J. Lab. Clin. Med. 115(1), 36-42 (1990)].
Elevated levels of TNF.alpha. are implicated in reperfusion injury, the inflammatory response which follows reperfusion, and is a major cause of tissue damage after blood flow loss [Vedder, et al., PNAS 87, 2643-2646 (1990)]. TNF.alpha. also alters the properties of endothelial cells and has various pro-coagulant activities, such as producing an increase in tissue factor pro-coagulant activity, suppressing the anticoagulant protein C pathway, and down-regulating the expression of thrombomodulin [Sherry, et al., J. Cell Biol. 107, 1269-1277 (1988)]. TNF.alpha. has pro-inflammatory activities which together with its early production (during the initial stage of an inflammatory event) make it a likely mediator of tissue injury in several important disorders including but not limited to, myocardial infarction, stroke and circulatory shock. TNF.alpha.-induced expression of adhesion molecules, such as intercellular adhesion molecules (ICAM) or endothelial leukocyte adhesion molecules (ELAM) on endothelial cells may be especially important [Munro, et al., Am. J. Path. 135(1), 121-132 (1989)].
It has been reported that TNF.alpha. is a potent activator of retrovirus replication including activation of HIV-1. [Duh, et al., Proc. Nat. Acad. Sci. 86, 5974-5978 (1989); Poll, et al., Proc. Nat. Acad. Sci. 87, 782-785 (1990); Monto, et al., Blood 79, 2670 (1990); Clouse, et al., J. Immunol. 142, 431-438 (1989); Poll, et al., AIDS Res. Hum. Retrovirus, 191-197 (1992)]. At least three types or strains of HIV (i.e., HIV-1, HIV-2 and HIV-3) have been identified. As a consequence of HIV infection, T-cell mediated immunity is impaired and infected individuals manifest severe opportunistic infections and/or unusual neoplasms. HIV entry into the T-lymphocyte requires T-lymphocyte activation. Other viruses, such as HIV-1, HIV-2 infect T-lymphocytes after T-cell activation. This virus protein expression and/or replication is mediated or maintained by this T-cell activation. Once an activated T-lymphocyte is infected with HIV, the T-lymphocyte must continue to be maintained in an activated state to permit HIV gene expression and/or HIV replication. Cytokines, specifically TNF.alpha., are implicated in activated T-cell mediated HIV protein expression and/or virus replication by playing a role in maintaining T-lymphocyte activation. Therefore, interference with cytokine activity such as prevention or inhibition of cytokine production, notably TNF.alpha., in an HIV-infected individual assists in limiting the maintenance of T-lymphocyte caused by HIV infection.
Monocytes, macrophages, and related cells, such as kupffer and glial cells, also have been implicated in maintenance of the HIV infection. These cells, like T-cells, are targets for viral replication and the level of viral replication is dependent upon the activation state of the cells. [Rosenberg, et al., The immunopathogenesis of HIV Infection, Advances in Immunology, 57 (1989)]. Cytokines, such as TNF.alpha., have been shown to activate HIV replication in monocytes and/or macrophages [Poli, et al., Proc. Natl. Acad. Sci., 87, 782-784 (1990)], therefore, prevention or inhibition of cytokine production or activity aids in limiting HIV progression for T-cells. Additional studies have identified TNF.alpha. as a common factor in the activation of HIV in vitro and have provided a clear mechanism of action via a nuclear regulatory protein found in the cytoplasm of cells [Osborn, et al., PNAS 86 2336-2340]. This evidence suggests that reducing TNF.alpha. synthesis may have an antiviral effect in HIV infections, by reducing transcription and thus virus production.
AIDS viral replication of latent HIV in T-cell and macrophage lines can be induced by TNF.alpha. [Folks, et al., PNAS 86, 2365-2368 (1989)]. A molecular mechanism for the virus inducing activity is suggested by TNF.alpha.'s ability to activate a gene regulatory protein (transcription factor, NF.kappa.B) found in the cytoplasm of cells, which promotes HIV replication through binding to a viral regulatory gene sequence (LTR) [Osborn, et al., PNAS 86, 2336-2340 (1989)]. TNF.alpha. in AIDS associated cachexia is suggested by elevated serum TNF.alpha. and high levels of spontaneous TNF.alpha. production in peripheral blood monocytes from patients [Wright, et al., J. Immunol. 141(1), 99-104 (1988)]. TNF.alpha. has been implicated in various roles with other viral infections, such as the cytomegalia virus (CMV), influenza virus, adenovirus, and the herpes family of viruses for similar reasons as those noted.
The nuclear factor .kappa.B (NF.kappa.B) is a pleiotropic transcriptional activator (Lenardo, et al., Cell 1989, 58, 227-29). NF.kappa.B has been implicated as a transcriptional activator in a variety of disease and inflammatory states and is thought to regulate cytokine levels including but not limited to TNF.alpha. and active HIV transcription [Dbaibo, et al., J. Biol. Chem. 1993, 17762-66; Duh, et al., Proc. Natl. Acad. Sci. 1989, 86, 5974-78; Bachelerie, et al., Nature 1991, 350, 709-12; Boswas, et al., J. Acquired Immune Deficiency Syndrome 1993, 6, 778-786; Suzuki, et al., Biochem. And Biophys. Res. Comm. 1993, 193, 277-83; Suzuki, et al., Biochem. And Biophys. Res Comm. 1992, 189, 1709-15; Suzuki, et al., Biochem. Mol. Bio. Int. 1993, 31(4), 693-700; Shakhov, et al., Proc. Natl. Acad. Sci. USA 1990,171, 35-47; and Staal, et al., Proc. Nati. Acad. Sci. USA 1990, 87, 9943-47]. Thus, it would be helpful to inhibit NF.kappa.B activation, nuclear translation or binding to regulate transcription of cytokine gene(s) and through this modulation and other mechanisms be useful to inhibit a multitude of disease states.
Many cellular functions are mediated by levels of adenosine 3',5'-cyclic monophosphate (cAMP). Such cellular functions can contribute to inflammatory conditions and diseases including asthma, inflammation, and other conditions (Lowe and Cheng, Drugs of the Future, 17(9), 799-807, 1992). It has been shown that the elevation of cAMP in inflammatory leukocytes inhibits their activation and the subsequent release of inflammatory mediators, including TNF.alpha. and NF.kappa.B. Increased levels of cAMP also lead to the relaxation of airway smooth muscle.
The primary cellular mechanism for the inactivation of cAMP is the breakdown of cAMP by a family of isoenzymes referred to as cyclic nucleotide phosphodiesterases (PDE) [Beavo and Reitsnyder, Trends in Pharm., 11, 150-155, 1990]. There are ten known members of the family of PDEs. It is well documented that the inhibition of PDE type IV (PDE 4) enzyme is particularly effective in both the inhibition of inflammatory mediator release and the relaxation of airway smooth muscle [Verghese, et al., Journal of Pharmacology and Experimental Therapeutics, 272(3), 1313-1320, 1995].
Decreasing TNF.alpha. levels and/or increasing cAMP levels thus constitutes a valuable therapeutic strategy for the treatment of many inflammatory, infectious, immunological, and malignant diseases. These include but are not restricted to: septic shock, sepsis, endotoxic shock, hemodynamic shock and sepsis syndrome, post ischemic reperfusion injury, malaria, mycobacterial infection, meningitis, psoriasis and other dermal diseases, congestive heart failure, fibrotic disease, cachexia, graft rejection, cancer, tumor growth, undesirable angiogenesis, autoimmune disease, opportunistic infections in AIDS, rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, other arthritic conditions, inflammatory bowel disease, Crohn's disease, ulcerative colitis, multiple sclerosis, systemic lupus erythrematosis, ENL in leprosy, radiation damage, and hyperoxic alveolar injury. Prior efforts directed to the suppression of the effects of TNF.alpha. have ranged from the utilization of steroids such as dexamethasone and prednisolone to the use of both polyclonal and monoclonal antibodies [Beutler, et al., Science 234, 470-474 (1985); WO 92/11383].
Angiogenesis, the process of new blood vessel development and formation, plays an important role in numerous normal and pathological physiological events. Angiogenesis occurs in response to specific signals and involves a complex process characterized by infiltration of the basal lamina by vascular endothelial cells in response to angiogenic growth signal(s), migration of the endothelial cells toward the source of the signal(s), and subsequent proliferation and formation of the capillary tube. Blood flow through the newly formed capillary is initiated after the endothelial cells come into contact and connect with a preexisting capillary. Angiogenesis is required for tumor growth beyond a certain size.
Inhibitory influences predominate in the naturally occurring balance between endogenous stimulators and inhibitors of angiogenesis [Rastinejad, et al., 1989, Cell 56:345-355]. In those rare instances in which neovascularization occurs under normal physiological conditions, such as wound healing, organ regeneration, embryonic development, and female reproductive processes, angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail.
Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases. A number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis [Moses, et al., 1991, Biotech. 9:630-634; Folkman, et al., 1995, N. Engl. J. Med., 333:1757-1763; Auerbach, et al., 1985, J. Microvasc. Res. 29:401-411; Folkman, 1985, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203; Patz, 1982, Am. J. Opthalmol. 94:715-743; and Folkman, et al., 1983, Science 221:719-725]. In a number of pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data suggests that the growth of solid tumors is dependent on angiogenesis [Folkman and Klagsbrun, 1987, Science 235:442-447].
The maintenance of the avascularity of the cornea, lens, and trabecular meshwork is crucial for vision as well as for ocular physiology. See, e.g., reviews by Waltman, et al., 1978, Am. J. Ophthal. 85:704-710 and Gartner, et al., 1978, Surv. Ophthal. 22:291-312. Currently, the treatment of these diseases, especially once neovascularization has occurred, is inadequate and blindness often results.
An inhibitor of angiogenesis could have an important therapeutic role in limiting the contributions of this process to pathological progression of the underlying disease states as well as providing a valuable means of studying their etiology. For example, agents that inhibit tumor neovascularization could play an important role in inhibiting metastatic and solid tumor growth.
Several kinds of compounds have been used to prevent angiogenesis. Taylor, et al. used protamine to inhibit angiogenesis, [Taylor, et al., Nature 297:307 (1982)]. The toxicity of protamine limits its practical use as a therapeutic. Folkman, et al. used heparin and steroids to control angiogenesis. [Folkman, et al., Science 221:719 (1983) and U.S. Pat. Nos. 5,001,116 and 4,994,443]. Steroids, such as tetrahydrocortisol, which lack gluco and mineral corticoid activity, are angiogenic inhibitors. Interferon .beta. is also a potent inhibitor of angiogenesis induced by allogeneic spleen cells [Sidky, et al., Cancer Research 47:5155-5161 (1987)]. Human recombinant interferon-.alpha. was reported to be successfully used in the treatment of pulmonary hemangiomatosis, an angiogenesis-induced disease [White, et al., New England J. Med. 320:1197-1200 (1989)].
Other agents which have been used to inhibit angiogenesis include ascorbic acid ethers and related compounds [Japanese Kokai Tokkyo Koho No. 58-131978]. Sulfated polysaccharide DS 4152 also shows angiogenic inhibition [Japanese Kokai Tokkyo Koho No. 63-119500]. A fungal product, fumagillin, is a potent angiostatic agent in vitro. The compound is toxic in vivo, but a synthetic derivative, AGM 12470, has been used in vivo to treat collagen II arthritis. Fumagillin and o-substituted fumagillin derivatives are disclosed in EPO Publication Nos. 0325199A2 and 0357061A1.
In U.S. Pat. No. 5,874,081, Parish teaches use of monoclonal antibodies to inhibit angiogenesis. In WO92/12717, Brem, et al. teach that some tetracyclines, particularly Minocycline, Chlortetracycline, Demeclocycline and Lymecycline are useful as inhibitors of angiogenesis. Brem, et al. teach that Minocycline inhibits angiogenesis to an extent comparable to that of the combination therapy of heparin and cortisone [Cancer Research, 51, 672-675, Jan. 15, 1991]. Teicher, et al. teach that tumor growth is decreased and the number of metastases is reduced when the anti-angiogenic agent of metastases is reduced when the anti-angiogenic agent Minocycline is used in conjunction with cancer chemotherapy or radiation therapy [Cancer Research, 52, 6702-6704, Dec. 1, 1992].
Macrophage-induced angiogenesis is known to be stimulated by TNF.alpha.. Leibovich, et al. reported that TNF.alpha. induces in vivo capillary blood vessel formation in the rat cornea and the developing chick chorioallantoic membranes at very low doses and suggested TNF.alpha. is a candidate for inducing angiogenesis in inflammation, wound repair, and tumor growth [Nature, 329, 630-632 (1987)].
All of the various cell types of the body can be transformed into benign or malignant tumor cells. The most frequent tumor site is lung, followed by colorectal, breast, prostate, bladder, pancreas, and then ovary. Other prevalent types of cancer include leukemia, central nervous system cancers, brain cancer, melanoma, lymphoma, erythroleukemia, uterine cancer, bone cancer, and head and neck cancer.
Cancer is now primarily treated with one or a combination of three types of therapies: surgery, radiation, and chemotherapy. Surgery involves the bulk removal of diseased tissue. While surgery is sometimes effective in removing tumors located at certain sites (e.g., in the breast, colon, and skin) surgery cannot be used in the treatment of tumors located in other areas (e.g., the backbone) nor in the treatment of disseminated neoplastic conditions (e.g., leukemia). Chemotherapy involves the disruption of cell replication or cell metabolism. Chemotherapy is used most often in the treatment of leukemia, as well as breast, lung, and testicular cancer.
Chemotherapeutic agents are often referred to as antineoplastic agents. The alkylating agents are believed to act by alkylating and cross-linking guanine and possibly other bases in DNA, arresting cell division. Typical alkylating agents include nitrogen mustards, ethyleneimine compounds, alkyl sulfates, cisplatin, and various nitrosoureas. A disadvantage with these compounds is that they not only attack malignant cells, but also other cells which are naturally dividing, such as those of bone marrow, skin, gastro-intestinal mucosa, and fetal tissue. Antimetabolites are typically reversible or irreversible enzyme inhibitors, or compounds that otherwise interfere with the replication, translation or transcription of nucleic acids. Thus, it would be preferable to find less toxic compounds for cancer treatment.
Matrix metalloproteinase (MMP) inhibition has been associated with several activities including inhibition of TNF.alpha. [Mohler, et al., Nature, 370, 218-220 (1994)] and inhibition of angiogenesis. MMPs are a family of secreted and membrane-bound zinc endopeptidases that play a key role in both physiological and pathological tissue degradation [Yu, et al., Drugs & Aging, 1997, (3):229-244; Wojtowicz-Praga, et al., Int. New Drugs, 16:61-75 (1997)]. These enzymes are capable of degrading the components of the extracellular matrix, including fibrillar and non-fibrillar collagens, fibronectin, laminin, and membrane glycoproteins. Ordinarily, there is a delicate balance between cell division, matrix synthesis, matrix degradation (under the control of cytokines), growth factors, and cell matrix interactions. Under pathological conditions, however, this balance can be disrupted. Conditions and diseases associated with undesired MMP levels include, but are not limited to: tumor metastasis invasion and growth, angiogenesis, rheumatoid arthritis, osteoarthritis, osteopenias such as osteoporosis, periodontitis, gingivitis, Crohn's disease, inflammatory bowel disease, and corneal epidermal or gastric ulceration.
Increased MMP activity has been detected in a wide range of cancers [Denis, et al., Invest. New Drugs, 15: 175-185 (1987)]. As with TNF.alpha., MMPs are believed to be involved in the invasive processes of angiogenesis and tumor metastasis.